Nipah virus (NiV) is the deadliest Paramyxovirus, with a mortality rate of up to 75% in humans. NiV and Hendra virus (HeV) (Henipaviridae genus) are emerging viruses within the Paramyxoviridae family, and their infections cause respiratory and encephalitic disease in humans and livestock. Entry of most paramyxoviruses requires the coordinated action of the attachment (HN, H, or G) and fusion (F) glycoproteins. The attachment protein (G for NiV) binds the cell receptor, and G-receptor-binding (ephrinB2 or B3 in the case of NiV) is thought to trigger the F protein. However, how F and G interactions link receptor binding to triggering and fusion-activation of the F protein remains poorly understood. Paramyxoviral F proteins exhibit canonical features common to class I fusion proteins, which cause fusion via a two-phase conformational cascade: the first phase progresses from a metastable pre-fusion state to a pre-hairpin intermediate (PHI), while the second phase is marked by transition from the PHI to a six-helix bundle hairpin, the final conformational change that leads to fusion of the viral and host cell membranes during viral entry. Our overall driving hypothesis is that fusion modulatory motifs in NiV-F and -G can manifest their effects through distinct intermediates of the fusion cascade. Our objective is to better understand the critical parameters that modulate the individual steps of the fusion cascade so as to facilitate the development of anti-viral therapeutics that target the entry process. For NiV, we recently developed a quantitative and kinetic F-triggering assay that can quantify the half-lives of distinct intermediates in the membrane fusion cascade. In addition, we have used a multi-faceted strategy to identify a receptor-induced conformational change in G critical to its ability to trigger F. We have developed sensitive cell-cell and virus-cell fusion kinetics assays to study NiV entry at less than BSL4 conditions, a new assay to detect F/G interactions, and novel conformational antibodies against F and G that helped us identify an allosteric F-triggering domain in G. These innovative and advantageous features allow us to use the Nipah virus system as an illuminative model for paramyxoviral entry. In addition, we have recently discovered novel determinants of fusogenicity in NiV-F and -G, including (a) a third heptad repeat (HR3) region in NiV-F that modulates membrane fusion, (b) the avidity of F and G interactions as a primary determinant of fusogenicity, and (c) specific domains in the head and stalk regions of G that are important for the allosteric triggering of F. We will use our innovative assays to study our novel determinants of fusogenicity. Thus, we propose the following two distinct but complementary aims: (1) To elucidate the novel fusion-modulatory role of the HR3 region in the NiV fusion cascade. (2) To understand the cooperativity between the F and G proteins during F triggering. The successful completion of these aims will not only facilitate the development of therapeutics that target entry of the deadly emerging NiV pathogen, but also advance our understanding of the parameters that govern NiV and paramyxovirus and class I viral membrane fusion and entry. PUBLIC HEALTH RELEVANCE: The paramyxoviruses include serious human pathogens, such as measles, mumps, human parainfluenza, Hendra, and Nipah viruses. The latter is the deadliest of the known paramyxoviruses. Understanding how Nipah virus infects cells, specifically the early events of membrane fusion and entry, will provide new approaches for prevention and therapy of not only Nipah virus, but more broadly for paramyxoviruses.